mmg_233_2013_genetics_genomicswikiaorg-20200214-history
Microbial Metagenomic Analysis and Comparison of Candlewood Lake Sediments in Winter Drawdown and Shallow Submerged Shoreline Areas
Candlewood Lake is the largest lake in Connecticut. It is a man-made lake that was created in 1928 for generation of hydroelectric power. Since the early 1980's, Eurasian watermilfoil has become a troublesome invasive species present in the lake. In an attempt to control the spread of this invasive plant, the Candlewood Lake Authority decided to implement a biennial deep drawdown (about 9-10 feet) of the lake during the winter months in an attempt to kill of the Eurasian milfoil which proliferates and thrives near the shoreline. During the years that there is not a deep drawdown, the lake is subjected to a shallow drawdown of approximately 4-5 feet. This biennial deep drawdown of the lake continues to this day and is the primary means of controlling the invasive plant Myriophillum spicatum. The Study To control the invasive plant Myriophillum spicatum (Eurasian watermilfoil) along the shores of Candlewood Lake, an annual water level drawdown is performed. As a result, unintended effects on microbial communities are possible. We performed metagenomic analysis and comparison of microbial communities in the sediment of the exposed drawdown shoreline region and the still submerged shoreline area to determine any impact on the lake’s microbiome. Looking at the Metagenome of Candlewood Lake Sediment In this two-pronged pilot study, environmental lake sediment samples were collected and metagenomic DNA was isolated and cleaned from the samples. Polymerase chain reaction (PCR) was used to amplify 16S rRNA genes. In the first part of the study, 16S rDNA PCR products were ligated to vectors, and several clones were chosen from each sample. The DNA sequences of these isolates were determined, and subsequently compared with known sequences in the BLAST database. In the second part of the study, community 16S rRNA genes from each sample were amplified with PCR for T-RFLP analysis. The terminal restriction fragments from each sample were compared. Methods DNA was isolated from the sediment samples, and 16S rDNA was amplified via PCR. DNA samples were purified and ligated to pDrive plasmid cloning vectors. Recombinant plasmids were transformed into electrocompetent E.coli. Transformed cells were plated, and after 48 hours ten submerged sample colonies and six drawdown sample colonies were selected for PCR. Colony PCR digests using HhaI and MspI were performed. Samples from the same colonies were also grown in LB broth, and plasmid DNA was isolated from the samples. Sample DNA concentrations were measured using a NanoDrop spectrophotometer. Based on these results, several samples that yielded sufficient DNA concentrations were chosen and sent to the Yale DNA Facility for DNA sequencing. DNA sequences were analyzed using BLAST and SDSC Biology Workbench. T-RFLP analysis was performed after amplification of 16S rRNA genes from the metagenomic samples. Fast cycling PCR was conducted, PCR products were cleaned, and restriction digestion was carried out using HhaI and MspI. After SureClean cleanup samples were sent to the Yale DNA Facility for fragment analysis. Candlewood Flowchart.jpg|Workflow for microbial metagenomic analysis of Candlewood Lake Sediment. BlastComparisons.jpg|BLAST COMPARISONS: 16S rRNA gene sequence alignments of cloned samples showed 98% or greater identity with a number of known sequences. Shown are representative examples of closest matches. A wide variety of aquatic and sediment environments are represented in these closely matching sequences. Textshade.jpg|TEXTSHADE results from SDSC Biology Workbench show degree of sequence alignment. Pairwise comparison.jpg|Pair wise comparisons of evolutionary distance as calculated by SDSC Biology Workbench. No clear grouping of submerged samples versus drawdown samples was shown. Identities.jpg|Identities computed with respect to DD5. No clustering of submerged and drawdown samples was apparent. CLUSTALWresults.jpg|CLUSTALW results: Tree shows possible evolutionary relationships between species based on 16S rRNA gene sequence similarity. TRFLP electropherogram.jpg|T-RFLP electropherogram results: Drawdown sample Hha1 (top left); Submerged sample Hha1 (bottom left); Drawdown sample MspI (top right); Submerged sample Msp1 (bottom right). Peaks for operational taxonomic units show similarity between HhaI digest samples and between MspI digest samples. Terminal Restriction Fragments.jpg|Number of Terminal Restriction Fragments present in drawdown (D) and Submerged (S) samples. Peaks from electropherograms that measured < 1% of total peak area were excluded. Findings Metagenomic analysis is a useful tool for detecting the presence of bacteria that are unable to be cultured in the laboratory (Davis, 2005). In this pilot study, such was the case demonstrated by much of our data, as many of the BLAST matches in our samples were found to be from different uncultured bacteria. This preliminary study did not show a clear difference between the metagenomes of shoreline Candlewood Lake samples taken from winter drawdown areas and areas that remain submerged year round. CLUSTALW tree results indicated that although several submerged sample 16S rRNA gene sequences were very similar to each other, they did not all cluster together apart from drawdown samples. Analysis of percent identities of the samples to each other indicated no grouping together of the submerged samples and the drawdown samples, supporting the CLUSTALW data. Additionally, BLAST matches showed both submerged and drawdown samples had similarity to genomes found in aquatic environments as well as in sediments and biofilms, suggesting there is no clear difference in the preferred microbiome of bacteria from either environment. T-RFLP results reinforced our preliminary conclusion. Electropherograms of the PCR-amplified MspI and HhaI digests showed similar populations in both the submerged and drawdown areas, although the relative abundance of the bacteria differed. There also appeared to be a number of OTUs in the submerged samples that were not present in the drawdown samples, which may indicate a different diversity of species in the underwater environment. Candlewood Lake is a manmade lake, created in 1928 for generation of hydroelectric power. Invasive weeds have been a fairly recent nuisance, and winter drawdowns of the water level have only been carried out since 1985 (Marsicano, 2009). These drawdowns have not been performed every year, and their level and duration varies from year to year. These varying conditions may be responsible for the absence of a separate, established winter biome on the shoreline. Additionally, the length of time required for populations of species with specific characteristics to evolve may need to be longer than just a winter season. Furthermore, when the winter drawdown water level is brought back up to summer levels, species from both environments can again mix freely, leading to a more uniform microbiome. Further study involving much larger sample sizes is needed to determine if there are any differences of statistical significance in the submerged and drawdown area microbiomes. References 1. Candlewood Lake Authority website. 2. Davis, K.E.R., S.J. Joseph, and P.H. Janssen. 2005. Effects of Growth Medium, Inoculum Size, and Incubation Time on Culturability and Isolation of Soil Bacteria. Applied and Enviro. Microb. ''71(2):826-834. 3. Labie, S.S. 1998. New soil sampling procedures and recommended EPA analytical methods. Memorandum, Florida Dept. of Environmental Protection. Marsicano, L. 2009. 4. Investigations into Eurasian Watermilfoil management by deep drawdown at Candlewood Lake. Candlewood Lake Authority. 5. Thies, J.E. 2007. Soil microbial community analysis using terminal restriction fragment length polymorphisms. ''Soil Science Society Am. J. 71:579-591.